Figure 5.

NM23 is a functional GEF for Rad, Gem, and Ras. (A) [α-³²P]GDP was prepared as described in Experimental Procedures. GST-Rad (1 μg each) was loaded with this [α-³²P]GDP (3 μCi each) at 37°C for 30 min, followed by incubation in the absence or presence of 1 μg of GST-nm23 and 10 mM ATP at 25°C for 5 min. The bound nucleotides were eluted and resolved by TLC. (B) One microgram each of GST-Rad, GST-Gem, and GST-Ras were loaded with 10 mM nonradioactive GDP at 37°C for 30 min, followed by UV irradiation. The protein was rebound to the GSH-Sepharose beads and was washed 3 times. The GDP-bound Rad was incubated in the presence of 10 μCi [γ-³²P]ATP and 1 μg of GST-nm23 at 25°C for 5 min, then was resolved by 12% SDS/PAGE. (C) One microgram of GST-Rad, GST-Gem, and GST-Ras were incubated with 3 μCi of [α-³²P]GDP prepared as described above at 37°C for 30 min. Samples were UV-irradiated for 30 min and were resolved by 12% SDS/PAGE. (D) GST-Rad-Sepharose was incubated with 10 mM nonradioactive GDP at 37°C for 30 min followed by 3 washes. Aliquots were incubated with 10 μCi [γ-³²P]ATP with or without 0.1 μg of GST-nm23 at 25°C for the time indicated. The beads were washed, and the nucleotides were eluted and resolved by TLC, and the amount of [γ-³²P]GTP was quantitated by using a PhosphorImager.